Introduction

Post-burn infections are considered as a major concern in burn centers. During the last years, Pseudomonas aeruginosa and Acinetobacter baumannii have been responsible for the most important hospital-borne infections and an emerging cause of antimicrobial treatment failure (Leseva et al. 2013; Aghamollaei et al. 2015; Ellis et al. 2015). These bacteria show an outstanding capacity to develop resistance against common antibiotics such as carbapenems, β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides through a wide variety of mechanisms. Therefore, demand to new and effective therapies is inevitable (Bonomo and Szabo 2006). In the last years, we encountered with an improved mortality in patients with burn infections in our burn hospital (Motahari Burn Center in Tehran). These infections were caused by the resistant bacteria, which were extensively resistant to almost all commonly used antibiotics and even high concentrations of silver and fluoride (Gholipourmalekabadi et al. 2016a). Therefore, the recent researches are focusing on the development of novel antibacterial agent for protection against such resistant bacteria. Among the antibacterial materials, some metallic cations such as silver and fluoride, and antimicrobial peptides (AMPs) showed promising results (Bakal et al. 2017; Chung and Khanum 2017; Gholipourmalekabadi et al. 2016a). Silver and silver-containing wound dressings are already used in protection of burn wounds against infections (Silver et al. 2006; Lachine et al. 2016). Antibacterial activity and cytotoxicity of silver components are concentration dependent (Gholipourmalekabadi et al. 2016a; Nezafati et al. 2012).

AMPs are one of the most promising compounds used as novel antimicrobial and therapeutic agents. AMPs are important members of the host defense system in eukaryotes with a broad ability to kill microorganisms including viruses, bacteria, protozoa, and fungi with different mechanisms such as disrupting the structure or the function of microbial cell membranes or interaction with ATP and directly inhibiting the action of certain ATP-dependent enzymes especially by cationic peptides (Amani et al. 2015). The cationic antibacterial peptides (containing 12–50 amino acids) can adopt amphipathic conformations with spatially separated hydrophobic and charged regions. The main advantage of such peptides, over other defensive molecules (i.e., antibiotics), is broad-spectrum antibacterial activity with rapid onset of killing and low levels of induced resistance compared with conventional antibiotics (Amani et al. 2015; Kosikowska and Lesner 2016). Accordingly, the CM11 peptide is one of cationic antimicrobial peptides that contains 11-residue sequence (WKLFKKILKVL-NH2) derived from cecropin A (residues 2–8) and from melittin residues 6–9 that have been designed and produced to improve the properties of parent peptides. Our recent studies showed high antimicrobial effects of CM11 against several human pathogenic bacteria (Moghaddam et al. 2012; Moghaddam et al. 2014; Amani et al. 2015; Azad et al. 2017).

Despite some promising results, the biocompatibility and non-cytotoxicity as well as antibacterial activity of silver and AMPs have still remained challenging (Gholipourmalekabadi et al. 2015b; Gholipourmalekabadi et al. 2016a; Albers et al. 2013; AshaRani et al. 2008; Pelillo et al. 2014). Considering the importance of burn infections caused by P. aeruginosa and A. baumannii, in this study we evaluated and compared antibacterial effects the CM11 peptide and 1% silver-doped bioactive glass against extensively drug-resistant (XDR) strains of P. aeruginosa and A. baumannii, which were isolated from burn patients. In this regard, we isolate XDR bacteria isolates from burn patients hospitalized at our burn center during the year 2016 and fully determine their antibiotic resistance patterns and also resistance mechanisms. Then, the effectiveness and safety of cationic antimicrobial peptide as a possible and reliable treatment strategy, versus 1% silver-doped bioactive glass (AgBG) against such XDR bacteria were evaluated both in vitro and in vivo.

Materials and methods

Bacterial identification, antibiotic susceptibility tests and resistance mechanisms

Seven P. aeruginosa and four A. baumannii were isolated from the wound exudate of burn patients admitted to the Burn Unit of Shahid Motahari Hospital in Tehran, Iran (2016). Nine bacterial standard strains were also included in the study. The specimens were identified using conventional biochemical tests (Hakemi Vala et al. 2014). All the bacteria isolated from burn patients were subjected to antimicrobial susceptibility testing against commonly used antibiotics (SM-Table 1) using disk diffusion method and minimum inhibitory concentration (MIC) according to clinical and laboratory standards institute (CLSI) guidelines (2007). E. coli ATCC 25922 and P. aeruginosa ATCC 27853 served as control strains.

The antibiotic resistance mechanisms studied in the current investigation were as follows: porin and efflux pump-associated resistances for P. aeruginosa, and beta-lactamase-associated resistance for both P. aeruginosa and A. baumannii (Bonomo and Szabo 2006). The clinical isolates were screened for metallo-beta-lactamase (MBL) production by combined disk diffusion test (CDDT) using imipenem (10 μg) and meropenem (10 μg) disks (Mast Group, Merseyside, UK) alone and in combination with ethylenediaminetetraacetic acid (EDTA) (Sigma).

Also, PCR was performed to detect the beta-lactamase genes such as blaIMP, blaVIM, and blaNDM in P. aeruginosa and blaIMP, blaVIM, blaNDM, blaOXA-23-like, blaOXA-51-like, blaOXA-24-like and blaOXA-58-like in A. baumannii. PCR condition and the primers sequences are shown in SM-Table 2 and SM-Table 3, respectively. Another resistance mechanism in P. aeruginosa clinical isolates is through efflux pump-associated resistance that was investigated by carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, ST. Louis, MO, USA) (Ardebili et al. 2014) and the expression level of mexD and mexA efflux pumps was determined by real-time PCR (RT-PCR) (Rotor-Gene 6000, Corbett Research, Germany) according to previously described protocols (Llanes et al. 2004). The list of the primers used in RT-PCR, their sequence and relevant product size are listed in SM-Table 4.

Porins are from other resistance mechanisms in P. aeruginosa clinical isolates. Porin gene from the clinical isolates was amplified by conventional PCR using oprD-specific primers (oprDF1 and oprDR1). The sequence of oprD gene was aligned and compared to that in P. aeruginosa PAO1 using multiple sequence alignment (http://multalin.toulouse.inra.fr/multalin/).

Preparation and characterization of CM11 peptide and 1% AgBG

Cationic antimicrobial peptide (CM11) was synthesized by a solid-phase synthesis method using p-methylbenzhydrylamine resin (Badosa et al. 2007; Moghaddam et al. 2014). The synthesized peptide was purified by reversed-phase semi-preparative HPLC on the C18 tracer column. 1% AgBG bioactive glass was synthesized by the sol–gel technique based on a [1 − (x + y)](58%SiO2–33%P2O5–9%CaO)–xCaF2–yAg2O system (Gholipourmalekabadi et al. 2016a).

Cellular responses to CM11 peptide and 1% AgBG

The effects of the various concentrations of peptide on cell viability and cytotoxicity of fibroblast L-929 cells and human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) were studied by MTT, LDH-specific activity assay and 4′-6-diamidino-2-phenylindole (DAPI) staining.

Cell isolation and culture

Fibroblast L-929 cells were purchased from Pasteur Institute of Iran and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 1% pen/strep, nystatin and amphotericin B, 2 mM Glutamax and 1 mM l-glutamine (all from Gibco, Carlsbad, CA, USA) and maintained in a humidified atmosphere of 5% CO2 at 37 °C. The mesenchymal stem cells were isolated from human adipose tissue obtained from patients undergoing elective liposuction surgery (Gholipourmalekabadi et al. 2016b).

Characterization of hAT-MSCs

The hAT-MSCs were identified by their fibroblast-like morphology and phenotype characteristics by light microscope and flow cytometry, respectively. The CD markers used for phenotypic characterizations of the cells were as Sca1, CD44, CD90 and CD105, CD11b, CD33, CD34 and CD45 (all obtained from eBioscience, Hatfield, Ireland, UK), selected based on the International Society for Cellular Therapy (ISCT) criteria (Dominici et al. 2006).

Cell viability, cytotoxicity and density

The effects of various concentrations (4 µg ml−1, 8 µg ml−1, 16 µg ml−1, 32 µg ml−1, 64 µg ml−1, 1 mg ml−1, 2 mg ml−1, 4 mg ml−1) of CM11 peptide and 1% AgBG on the cell viability and cytotoxicity were determined by MTT and LDH assays, respectively. For this purpose, the cells were exposed to peptide and AgBG for 24, 48 and 72 h. MTT and LDH assays were carried out as described earlier (Gholipourmalekabadi et al. 2015a; Rostami et al. 2015). The density of the cells after treatment with peptide and 1% AgBG was determined by staining the cells with DAPI. The average number of the cells per high-power microscope field (HPF, n = 10; ×40 objective) was determined and compared with control.

Bacterial susceptibility to CM11 peptide and 1% AgBG

MIC and MBC assays

The MIC of peptide and 1% AgBG for all clinical isolates and bacterial standard strains was determined by microdilution broth method, according to clinical and laboratory standards institute (CLSI) guidelines (2007). Serial twofold dilutions for peptide and 1% AgBG (4 µg ml−1, 8 µg ml−1, 16 µg ml−1, 32 µg ml−1, 64 µg ml−1, 1 mg ml−1, 2 mg ml−1 and 4 mg ml−1) were prepared in cation-adjusted Mueller–Hinton broth (CAMHB). MBC was defined as the lowest concentration of peptide to have at least 99.9% killing of the initial inoculum.

In vivo biocompatibility of CM11 peptide and 1% AgBG

The biocompatibility of the CM11 peptide and 1% AgBG was assessed at 3 and 7 days after subcutaneously injection of various concentrations of peptide and 1% AgBG (ranging from 4 µg ml−1 to 4 mg ml−1) in BALB/c mice (Rostami et al. 2015). The average number of host inflammatory cells infiltrated into injected site was recorded and compared between the experimental groups. The average number of the cells of 10 high-powered field (HPF) was reported.

Results and discussion

The methodology and results of the current study are illustrated in Fig. 1.

Fig. 1
figure 1

Schematic of the current study. The bacteria were isolated from exudate of patients with burn wounds and characterized. Extensively drug-resistant (XDR) bacteria as well as standard strain bacteria were subjected to this study. Cationic antimicrobial peptide (CM11) and 1% silver-doped bioactive glass (1% AgBG) were synthesized. Antibacterial activity, in vitro and in vivo biocompatibility and safety of peptide and 1% AgBG were evaluated and compared between experimental groups

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Bacterial identification, antibiotic susceptibility tests and resistance mechanisms

Seven antibiotic-resistant P. aeruginosa and four antibiotic-resistant A. baumannii strains were isolated from wound exudate of the burn patients. Antibiogram results revealed that all eleven isolates were extensively drug resistant (XDR), MBL producing and sensitive to Colistin (Fig. 2a). The results also showed that three clinical isolates (two P. aeruginosa and one A. baumannii) were resistant to 1% AgBG disk. The results obtained from this work reveal that silver may not be able to protect burn patients against the MDR bacteria and should be replaced with another efficient antibacterial agent.

Fig. 2
figure 2

Bacterial resistance tests. a Antibiogram of commonly used antibiotics and 1% AgBG for P. aeruginosa and A. baumannii. All P. aeruginosa clinical isolates were sensitive to Colistin (Col). All clinical isolates were resistant to 1% AgBG. b Resistance mediated by beta-lactamase. All isolates were positive for MBL (CDDT test). (i) IMI 10 µg disk; (ii) IMI10 µg + EDTA 10 µg disk; (iii) MEM disk; (iv) MEM 10 µg + EDTA 10 µg disk. PCR products of blaIMP genes in P. aeruginosa isolates visualized in gel electrophoresis. Lane M: 100 bp DNA size marker; Lane P: positive control; Lane N: negative control; Lane 1–3: blaIMP positive P. aeruginosa isolates. Multiplex PCR products of blaOXA-24-like, blaOXA-23-like, and blaOXA-51-like genes in A. baumannii isolates visualized in gel electrophoresis. Lane M: 100 bp DNA size marker; Lane P: positive control; Lane N: negative control; Lane 1–3: blaOXA-23-like, blaOXA-51-like and blaOXA-24-like genes positive isolates. c΄ Relative expression of mexD and mexA in P. aeruginosa clinical isolates. c΄΄ Gel electrophoresis analysis of the RT-PCR products. Lane M: DNA size marker; Lane P: positive control; Lane N: negative control; Lane 1–7: mexD-positive isolates. Gel electrophoresis analysis of the RT-PCR products. Lane M: DNA size marker; Lane P: positive control; Lane N: negative control; Lane 1–3: mexA-positive isolates. d PCR products of OprD gene. The PCR products were run on 1.5% agarose gels, the corresponding bands were characterized and their sizes were compared to that in positive sample (P. aeruginosa PAO1). Lane M: GeneRuler 100 bp DNA Ladder (Sinaclon, Iran); Lane N: negative control; Lane 1 and 3: oprD gene-positive isolates: Lane 2 and 4: oprD + IS gene with insertion sequence

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The MIC results indicating the resistance of clinical isolates to these antibiotics are shown in Table 1. The seven P. aeruginosa and four A. baumannii isolates were highly resistant to carbapenem, with imipenem and meropenem MICs of ≥ 32 μg/ml. The carbapenem-resistant isolates were also resistant to ciprofloxacin and ceftazidime with MICs of ≥ 64 μg/ml. These findings indicate that carbapenem may be no longer the “gold standard” for the treatment of the infections caused by MDR bacteria isolated from burn patients.

Table 1 Minimum inhibitory concentrations (MIC µg ml−1) of different antibiotics against the bacteria isolated from hospitalized burn patients at Shahid Motahari Hospital, Tehran, Iran
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Metallo-β-lactamases are one of the most important factors responsible for increasing prevalence of Gram-negative bacterial resistance. Despite the worldwide rise of metallo-β-lactamases (MBLs), carbapenem resistance mediated by MBLs is common in Iran (Noori et al. 2014). Resistance mediated by MBLs, able to hydrolyze all β-lactams except monobactams, is an emerging problem among P. aeruginosa isolates in many countries of Asia, Europe, Africa, and South America (Fallah et al. 2012). As shown in Fig. 2b (disk diffusion), all P. aeruginosa and A. baumannii isolates were MBL producers.

The presence of β-lactamase genes is shown in SM-Table 5, SM-Table 6 and Fig. 2b. All A. baumannii isolates were positive for blaOXA-23-like, blaOXA-51-like and blaOXA-24-like and negative for blaOXA-58-like β-lactamases (SM-Table 6 and Fig. 2b). All P. aeruginosa and two A. baumannii isolates were positive for blaIMP, while blaVIM and blaNDM genes were not observed in all clinical isolates (SM-Table 5, SM-Table 6).

Carbonyl cyanide m-chlorophenylhydrazone (CCCP) is an efflux pump inhibitor (EPI) that indirectly inhibits efflux pump through disrupting the proton motive force and subsequently facilitating proton transfer across the bacterial membrane (Ikonomidis et al. 2008). According to the CCCP results, MIC value for ciprofloxacin with CCCP showed more than fourfold reduction (in three isolates of A. baumannii and five isolates of P. aeruginosa) and eightfold reduction (in one isolate of A. baumannii and two isolates of P. aeruginosa) in comparison to MIC values of ciprofloxacin without CCCP. These findings indicate that the increased activity of efflux pumps in clinical isolates may be one of the main causes of their resistance to ciprofloxacin.

The relative expression of mexD and mexA efflux pump genes in seven P. aeruginosa isolates was determined by RT-PCR and the results were compared with those in P. aeruginosa PAO1, as control sample (Fig. 2c). The gene expression analysis showed an increased relative expression of mexD (1.3–6.77-fold) and mexA (1.1–54.9-fold) in all P. aeruginosa clinical isolates when compared to those in P. aeruginosa PAO1 (p ≤ 0.05) (Fig. 2c΄and c΄΄), indicating the important role of mexD and mexA in resistance of P. aeruginosa isolated from burn patients against antibiotics.

Insertional inactivation through insertion sequence (IS) elements is one of the most common mechanisms associated with lack of function of OprD (Evans and Segal 2007). According to our results (Fig. 2d), only two isolates showed an increased PCR product size when compared with P. aeruginosa PAO1 (control). According to the data obtained from the multiple sequence alignment analysis of the IS-positive isolates and IS finder database nomenclature (https://www-is.biotoul.fr), these bacteria carry ISPpu21 and ISPa1328 elements in their oprD gene.

Cationic AMP characterization

HPLC and electrospray ionization mass spectrometry were used for purification and to confirm peptide identity, respectively. For HPLC, the active fraction was loaded on C18 tracer column using a linear gradient from 10 to 60% acetonitrile in water with 0.1% TFA over 50 min. The active peak corresponds to the peptide control obtained with > 95% HPLC purity. Also, the molecular mass of peptide was determined to be 1415.85 Da by electrospray ionization mass spectroscopy (SM-Fig. 1A and SM-Fig. 1B).

Cell’s responses to CM11 peptide and 1% AgBG

MSC characterization

The morphology of the cells during the expansion in flask was observed under light microscope (SM-Fig. 2A). The AT-MSCs showed a spindled shape similar to fibroblast morphology. The phonotypic characterization of the isolated cells was performed by flow cytometry. The flow cytometry result is shown in SM-Fig. 2B. 98.5% and 97.9%, 98.4 and 96.5% of the isolated cells were positive for Sca1, CD44, CD90 and CD105, respectively, while the cells were almost negative for CD11b, CD31, CD34 and CD45. These results indicate that more than 95% of the isolated cells were identified as mesenchymal stem cells (Mohammadzadeh et al. 2014).

Stem cell viability and cytotoxicity

The safety of novel antimicrobial agents against human cells is critical for minimizing their side effects. High cytotoxicity of such agents profoundly limits their applications in clinic (Bem et al. 2014; Ramandi et al. 2017; Moravej et al. 2018). For example, the most important complication associated with the use of silver as an antimicrobial agent is its strong biocidal property (Gholipourmalekabadi et al. 2015b; Nezafati et al. 2012). Furthermore, silver retards wound healing and causes formation of hypertrophic scar (Qian et al. 2017).

The effects of various concentrations (ranging from 4 µg ml−1 to 4 mg ml−1) of peptide versus 1% AgBG on the AT-MSC and fibroblast viability and cytotoxicity were determined by MTT and LDH (Figs. 3 and 4). At 24-h post-incubation for both AT-MSC and fibroblast samples, no difference in cell viability and cytotoxicity levels was observed in 64 µg ml−1 of CM11 peptide and 1% AgBG (p ≥ 0.05). At 48-h exposure time interval, both CM11 peptide and 1% AgBG exhibited cytotoxicity effects at concentrations of ≥ 2 mg ml−1 and 64 µg ml−1, respectively (p ≤ 0.05). After 72 h, 1% AgBG negatively affected the cell viability of the cells in concentrations of ≥ 32 µg ml−1 (p ≤ 0.05), while at the same time, CM11 peptide in concentrations of ≤ 1 mg ml−1 showed no significant cytotoxicity against the tested cells (p ≥ 0.05). The results also indicated that AT-MSCs were a little more sensitive than fibroblast when exposed to peptide and 1% AgBG. The results revealed that the cytotoxicity effect of peptide was less than 1% AgBG. In addition, it is estimated that the cytotoxicity exhibition of 1% AgBG after 72 h will be greatly more than peptide due to the sustained release of silver from bioactive glass. All the findings obtained from MTT and LDH were confirmed by the DAPI staining of the cells and determination of the cell density.

Fig. 3
figure 3

The effects of various concentrations of CM11 peptide on viability, cytotoxicity and density of AT-MSCs (a) and fibroblasts (b). The cell nuclei were stained with DAPI and counted. *Significant difference with control (p ≤ 0.05)

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Fig. 4
figure 4

The effects of various concentrations of 1% AgBG on viability, cytotoxicity and density of AT-MSCs (a) and fibroblasts (b). The cell nuclei were stained with DAPI and counted. *Significant difference with control (p ≤ 0.05)

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MIC and MBC of CM11 peptide and 1% AgBG

Amphipathic cationic peptides such as the CM11 act as a cell-permeable agent. Their positive charge causes electrostatic interactions with the poly-anionic compounds in the surface of bacterial cell membranes including lipopolysaccharide in Gram-negative bacteria and teichoic acid in Gram-positive bacteria. Their hydrophobic region enables them to penetrate the cell membrane, which leads to hole formation, resulting in the leakage of essential cellular components or passage of hydrophobic compounds across the cell membrane (Shai 2002; Nguyen et al. 2011). Based on the studies reported by Shai and Ferre et al., CM11 peptide kills bacteria via a “carpet-like” mechanism. Accordingly, after carpeting and thinning of the bacteria cell membrane by peptide, at a critical threshold concentration, peptide forms toroidal transient holes in the membrane and above this concentration, the membrane disintegrates and forms micelles after disruption of the bilayer curvature (Shai 2002; Ferre et al. 2006). In general, the damaging property of AMPs depends on charge, hydrophobicity, amphipathicity, stereochemistry, and propensity of peptides to form barrels. In addition, sensitivity of eukaryotic cells to AMPs also depends on variations in membrane lipid compositions and membrane hydrophobicity. Silver ions show toxicity activity through degradation of essential molecules such as DNA and RNA (Feng et al. 2000; Jeon et al. 2003).

The MIC and MBC values of peptide and 1% AgBG for standard strains, and A. baumannii and P. aeruginosa isolates are shown in Tables 2 and 3. The results showed that CM11 peptide and 1% AgBG had MIC of ≥ 8 μg ml−1 and ≥ 1 mg ml−1 for bacterial standard strains, respectively. MBC of peptide and 1% AgBG for bacterial standard strain was ≥ 8 μg ml−1 and ≥ 2 mg ml−1, respectively (Table 2). Peptide and 1% AgBG had MIC of ≥ 16 μg ml−1 and ≥ 4 mg ml−1 for clinical isolates, respectively. In addition, MBC of peptide and 1% AgBG for these resistant bacteria was ≥ 32 μg ml−1 and ≥ 4 mg ml−1, respectively (Table 3). Among the clinical isolates, two P. aeruginosa and one A. baumannii were resistant to 1% AgBG even in 4 mg ml−1.

Table 2 MIC and MBC of CM11 peptide and 1% AgBG for bacterial standard strains
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Table 3 MIC and MBC of CM11 peptide and 1% AgBG for clinical isolates
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Our results clearly indicate that some of XDR bacteria isolated from burn patients are resistant to all antibiotics and even high masses of silver. On the other hand, the CM11 peptide in concentrations of ≤ 1 mg ml−1 did not affect the viability of both AT-MSCs and fibroblasts and showed high biocompatibility property, while the safe concentrations of 1% AgBG for AT-MSCs and fibroblasts were ≤ 16 μg ml−1. Taken together, our interesting findings reveal that unlike 1% AgBG, peptide has an efficient antibacterial activity, even for XDR clinical isolates, in its safe concentration. These data may alter the current treatment strategies that use 1% silver sulfadiazine or skin substitute transplantation with non-/weak antibacterial property and suggest that peptide therapy as a potential candidate, reliable, alternative and safe strategy can be used for the treatment of burn infections caused by multidrug-resistant bacteria.

Accordingly, Huang et al. (2013) showed that antimicrobial peptide Epinecidin-1 protects skin wounds created in mice against methicillin-resistant Staphylococcus aureus. It has also been reported the synergic anti-mycobacterial activity of cationic a-helical peptides with rifampicin (Khara et al. 2014). Immuno-modulatory and broad-spectrum antibacterial property of the antimicrobial peptide piscidin were reported by Chen et al. (2015). Non-cytotoxicity effects of antimicrobial proline-rich peptide for macrophages have been shown by Pelillo et al. (2014).

In vivo biocompatibility

The high biocompatibility of antibacterial agents is very critical and guarantees their safety. The body immune responses after subcutaneous injection of various concentrations of both peptide and 1% AgBG were investigated and the results are shown in Fig. 5. Our findings showed that CM11 peptide did not stimulate body immune system by both 3 and 7 days post-injection, as infiltration of LC, MQ and PC did not change in this group compared with control (the animals subcutaneously injected with PBS). In 1% AgBG samples, the average number of LC and MQ was significantly increased in concentrations of ≥ 64 µg ml−1 by day 3. At day 7 post-injection of 1% AgBG, the average number of LC and MQ in concentrations of ≥ 64 µg ml−1 remained significantly increased. In this time, the average number of PC in concentrations of ≥ 16 µg ml−1 was significantly higher than those in control sample. In our previous studies (Gholipourmalekabadi et al. 2016a; Nezafati et al. 2012), we showed that AgBG has a durable antibacterial activity through a sustained release of silver ions. In those studies, AgBG showed concentration-dependent antibacterial activity. In higher concentrations, silver shows cytotoxicity for eukaryote cells, as reported by many studies (Gholipourmalekabadi et al. 2016a; Nezafati et al. 2012; Franci et al. 2015). Our findings indicate that CM11 peptide has high biocompatibility and safety in all evaluated concentrations, while biocompatibility of 1% AgBG is highly dependent on its concentration.

Fig. 5
figure 5

In vivo biocompatibility. Various concentrations of CM11 peptide and 1% AgBG (ranging from 4 µm ml−1 to 4 mg ml−1) were injected subcutaneously. At 3- and 7-day post-injection interval times, infiltration of inflammatory cells was counted and reported. *Significant difference with control (p < 0.05)

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Conclusion

Increasing incidence of extensively drug-resistant bacteria isolated from burn patients has become a serious problem in burn centers. In this study, we showed that clinical isolates are extensively resistant to commonly used antibiotic and 1% silver-doped bioactive glass. On the other hand, cationic antimicrobial peptide (CM11) shows to be an effective and safe antibacterial agent for the possible treatment of the infections caused by these bacteria. Our interesting results may attract the attention of many researchers in this area for clinical use of cationic AMPs such as CM11 in health centers as a prevention/treatment strategy of extensively drug-resistant bacterial infections.